Reversible protein phosphorylation is one of the most common mechanisms for regulating cell behavior in eukaryotes. Genome sequencing efforts have uncovered hundreds of new predicted protein kinases. For most, we have little insight into their biological function: protein substrates have been identified for only a minority, and pathways that control their activation are generally obscure. Targeted proteome-wide biochemical screens have great utility in rapidly providing functional information regarding sizable protein families such as protein kinases. We propose to systematically investigate the substrate specificity of each of the 124 protein kinases from Saccharomyces cerevisiae. We will accomplish this by adapting our recently described peptide library method to enable high throughput analysis. Each kinase will be overexpressed in yeast, purified, and subjected to peptide library screening to determine its consensus phosphorylation motif. We will identify candidate substrates for every kinase through protein sequence database searching and integration with other kinase-directed yeast proteomics efforts. We will validate target substrates of selected kinases by substantiating their sites of phosphorylation in vivo, making use of motif-directed phosphoprotein detection and affinity reagents. This work will contribute to our fundamental understanding of how specificity is achieved by protein kinases and identify critical connections in signaling networks. In addition, we will provide a general resource for researchers studying signal transduction and phosphorylation-dependent regulation of proteins.